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Objectives: Early embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal-to-zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high- or low- competence mouse strains., Materials and Methods: In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real-time quantitative PCR (RT-qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment., Results: The fertilization and 2-cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty-nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT-qPCR. Prkce knockout mice showed a reduced 2-cell rate (Prkce +/+ 80.1% vs. Prkce -/- 32.4%) that could be rescued by Prkce cRNA injection (2-cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation., Conclusions: The transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2-cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high-quality mature oocytes., (© 2022 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Cryopreservation techniques for mammalian oocytes and embryos have rapidly progressed during the past two decades, emphasizing their importance in various assisted reproductive technologies. Pregnancies and live births resulting from cryopreserved oocytes and embryos of several species including humans have provided proof of principle and led to the adoption of cryopreservation as an integral part of clinical in vitro fertilization. Considerable progress has been achieved in the development and application of the cryopreservation of mammalian oocytes and embryos, including preservation of the reproductive potential of patients who may become infertile, establishment of cryopreserved oocyte banks, and transport of oocytes and embryos internationally. However, the success rates are still far lower than those obtained with fresh oocytes and embryos, and there are still obstacles that need to be overcome. In this review, we address the major obstacles in the development of effective cryopreservation techniques. Such knowledge may help to eliminate these hurdles by revealing which aspects need improvement. Furthermore, this information may encourage further research by cryobiologists and increase the practical use of cryopreservation as a major part of assisted reproductive technologies for both humans and animal species.

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.

The development capability of reconstructed bovine embryos via ovum pick-up (OPU)-somatic cell nuclear transfer (SCNT) technique has been influenced by the maternal lineage of oocyte cytoplasm, but the underlying mechanism remains unclear. Since mitochondria are the richest maternal-inherited organelle, in this study, we intended to clarify the effect of mtDNA haplotypes on cloning efficiency. By PCR-RFLP method, we identified mtDNA haplotypes A and B, differing in six restriction sites. Reconstructed embryos with haplotype A cytoplast achieved better fusion and blastocyst formation rate (64.6% and 39.4%), as compared with haplotype B (53.6% and 26.3%; P < 0.05). To further evaluate the role of mitochondria, the quantity of mtDNA, ATP content, and mRNA level of mtDNA-encoded COXI, COXIII in both oocytes were measured. Our data indicated that mtDNA copy number in haplotype A oocyte was significantly higher than that in haplotype B oocyte, both at the GV (10(5.03 +/- 0.69) vs. 10(4.81 +/- 0.86) copies/oocyte) and MII stages (10(5.31 +/- 0.71) vs. 10(5.13 +/- 0.63) copies/oocyte; logarithmically transformed values; P < 0.05). ATP content in type A oocyte was also greater at the GV (1.67 +/- 0.09 vs. 1.27 +/- 0.1 pmol) and MII stages (5.18 +/- 0.07 vs. 2.68 +/- 0.03 pmol; P < 0.05). Similarly, the mRNA expression level of mtDNA-encoded COXI and COXIII in haplotype A oocyte was significantly higher comparing to haplotype B oocyte (3.3 +/- 2.0 x 10(3) vs. 0.68 +/- 0.45 x 10(3); 24.9 +/- 10.5 x 10(3) vs. 9.4 +/- 3.3 x 10(3), respectively; P < 0.05). The data suggest that mitochondrial structure, quantity, and function may significantly affect the developmental competence of reconstructed embryos., ((c) 2007 Wiley-Liss, Inc.)

Basonuclin is a zinc-finger protein found in abundance in oocytes. It qualifies as a maternal-effect gene because the source of pre-implantation embryonic basonuclin is maternal. Using a transgenic-RNAi approach, we knocked down basonuclin specifically in mouse oocytes, which led to female sub-fertility. Basonuclin deficiency in oocytes perturbed both RNA polymerase I- and II-mediated transcription, and oocyte morphology was affected (as evidenced by cytoplasmic and cell surface abnormalities). Some of the affected oocytes, however, could still mature to and arrest at metaphase II, and be ovulated. Nevertheless, fertilized basonuclin-deficient eggs failed to develop beyond the two-cell stage, and this pre-implantation failure accounted for the sub-fertility phenotype. These results suggest that basonuclin is a new member of the mammalian maternal-effect genes and, interestingly, differs from the previously reported mammalian maternal-effect genes in that it also apparently perturbs oogenesis.

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte's response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach.

The paucity of biological material has inhibited identifying genes that are differentially expressed during mammalian oogenesis and preimplantation development. We report here the linear amplification of mRNA from small numbers of mouse oocytes and preimplantation embryos to generate amounts of sense RNA that are sufficient for suppression subtractive hybridization. The resulting oocyte-specific and 8-cell-specific cDNA libraries were partially characterized, and the known oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1(oo) genes were found in the oocyte-specific cDNA library but not in the 8-cell-specific library. Further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries should furnish a trove of information regarding temporal changes in gene expression during oogenesis and preimplantation development in the mouse.